Fig. 3.

Suppression of SNAP-induced sGC activity via iNOS-mediated nitrosylation of sGC. Longitudinal muscle cells isolated from colons of control and TNBS-treated mice (A) or from muscle strips cultured in the presence of IL-1β (10 ng/ml) (B) or TNF-α (1 nM) (C) for 48 hours were treated with different concentrations of SNAP, and sGC activity was measured as described in Materials and Methods. In some experiments, muscle strips were cultured in the presence of IL-1β (10 ng/ml) (B) or TNF-α (1 nM) (C) plus iNOS inhibitor 1400W (10 µM) for 48 hours. Basal sGC activity was not significantly different between control and TNBS-treated mice (2.61 ± 0.32 versus 2.54 ± 0.36 pmol/mg protein) or between control and cytokine-treated muscle strips (2.48 ± 0.36 versus 2.62 ± 0.36 pmol/mg protein). Values are the means ± S.E.M. of five experiments. **P < 0.01, significant inhibition of SNAP-induced sGC activity. (D) Representative immunoblot of four to five different experiments. Longitudinal muscle cells isolated from colons of control and TNBS-treated mice or from muscle strips cultured in the presence of IL-1β (10 ng/ml) or TNF-α (1 nM) for 48 hours. In some experiments, muscle strips were cultured with IL-1β (10 ng/ml) or TNF-α (1 nM) in the presence of iNOS inhibitor 1400W (10 µM) for 48 hours. Lysates were used to measure iNOS expression and S-nitrosylation of sGC (sn-sGC) as described in Materials and Methods.